WO2008142619A1

WO2008142619A1 - Use of herbal compositions in the protection and enhancement of extracellular matrix components

Info

Publication number: WO2008142619A1
Application number: PCT/IB2008/051915
Authority: WO
Inventors: Gabriel Nussbaum; William Zev Levine
Original assignee: Izun Pharmaceuticals Corp
Current assignee: Izun Pharmaceuticals Corp
Priority date: 2007-05-17
Filing date: 2008-05-15
Publication date: 2008-11-27
Anticipated expiration: 2009-11-17

Abstract

The present invention provides a method for treating and/or preventing degeneration of collagen and/or other extracellular matrix components in a mammalian subject, wherein said method comprises administering to said subject a herbal composition comprising an extract of Centella asiatica and/or an extract of Sambucus nigra as the active component (s).

Description

Use of herbal compositions in the protection and enhancement of extracellular matrix components

Field of the invention

The present invention relates to the use of herbal formulations containing either single herbal extracts or mixtures of different herbal extracts, for the protection and enhancement of extracellular matrix (ECM) components such as collagen and hyaluronic acid. The present invention is useful for the treatment of conditions that would benefit from increased production or strength of the extracellular matrix such as in degenerative conditions of the joints, skin, or mucosa, and wound healing, and for cosmetic applications.

Background of the invention

The ECM is composed of various types of glycoproteins, proteoglycans and polysaccharides. The ECM is crucial for supporting and anchoring cells and sequestering the growth factors that act upon them. Intercellular communication is therefore very much dependent upon an intact and healthy ECM.

Collagens are among the most abundant glycoproteins in the ECM and confer strength and integrity to the connective tissue.

Hyaluronic acid is a polysaccharide that forms a major constituent of the ECM of connective tissue. Hyaluronic acid is also found in large amounts in the synovial fluid of joints, and the vitreous humor of the eyes. Among its many functions it is known as a binding, lubricating, and protective agent. Degenerative conditions affecting the joints are a major cause of morbidity. Breakdown of the integrity of the synovial fluid and connective tissue elements in the joints are a major component of these conditions regardless of their etiology. Therapeutic agents that protect synovial fluid and connective tissue elements are therefore expected to be of benefit for the treatment of these conditions.

Similarly, the eyeball between the retina and the lens is bathed in a clear gelatinous substance known as the vitreous humor, of which collagen and hyaluronic acid are important components. Agents that enhance these factors would be of benefit for acute and chronic conditions affecting the integrity of the eye.

Skin changes with age, such as wrinkles, color changes and changes in tone are a major cosmetic concern. Given its ability to strengthen and give fullness to the connective tissue, agents that build or protect ECM components, in particular collagen and hyaluronic acid, are expected to benefit in the management of these changes .

It is a purpose of the present invention to provide herbal compositions and methods of using same in the management of conditions characterized by breakdown or loss of integrity of collagen and other ECM components such as hyaluronic acid.

A further purpose of the present invention is to provide a cosmetic composition containing certain herbal extracts as the cosmetically-active ingredients. Further objects and advantages of the present invention will become apparent as the description proceeds.

Summary of the invention

The inventors have now unexpectedly found that compositions comprising an extract of Centella asiatica alone, an extract of Sambucus nigra alone, or a mixture of extracts of Sambucus nigra, Centella asiatica and Echinacea species are effective in the treatment of conditions characterized by degeneration of collagen and certain other extracellular matrix components such as hyaluronic acid. Examples of such conditions include degenerative joint diseases such as those caused by arthritis (osteoarthritis or rheumatoid arthritis), ocular conditions such as keratoconjunctivitis, skin conditions such as age related changes such as wrinkles, and wound healing of all types .

The present invention is thus primarily directed to a method for treating and/or preventing degeneration of collagen and/or other extracellular matrix components (in particular, hyaluronic acid) , wherein said method comprises administering a herbal composition comprising an extract of Centella asiatica and/or Sambucus nigra as the active component (s) to a mammalian subject in need of such treatment. In one preferred embodiment, the mammalian subject is a human subject.

In addition to the two aforementioned herbal extracts, the composition used in the present invention may also comprise extracts of additional plants. Such additional plants may include any of the plants commonly used in herbal remedies. A preferred additional plant is a plant of the Echinacea genus. In one preferred embodiment, the method comprises the administration of a herbal composition comprising a mixture of extracts of plants selected from the group consisting of Sarnbucus nigra, Centella asiatica and one or more Echinacea species. In a particularly preferred embodiment, the method involves the use of a composition containing the aforementioned three plant extracts, wherein said composition is the mixture of Sambucus nigra, Centella asiatica and Echinacea purpurea that is described in co-owned international patent application no. PCT/IL02/00402 (published as WO 02/094300), the disclosure of which is incorporated herein in its entirety.

In another preferred embodiment, the method involves the administration of a composition containing Centella asiatica as the sole active ingredient.

In a further preferred embodiment, the method involves the administration of a composition containing Sambucus nigra as the sole active ingredient .

In another aspect, the present invention is also directed to the use of a herbal composition comprising an extract of Centella asiatica and/or an extract of Sambucus nigra as the active component (s) in the preparation of a medicament and/or cosmetic agent for treating and/or preventing degeneration of collagen and/or other extracellular matrix components. The herbal composition, and the medical and cosmetic indications for the use of the medicament and/or cosmetic agent are as disclosed hereinabove (in relation to the method of treatment) and as described in more detail hereinbelow.

The present invention further provides a composition suitable for cosmetic use comprising an extract of Centella asiatica and/or an extract of Sambucus nigra as the cosmetically-active component ( s ). In a preferred embodiment, said composition comprises a mixture of extracts of Centella asiatica, Sambucus nigra and an Echinacea species (such as Echinacea purpurea) .

The present invention is also directed to topical dosage forms containing the above-disclosed cosmetic composition, wherein said dosage form is particularly suitable for delivering said composition to the dermal and sub-dermal tissues.

All the above and other characteristics and advantages of the present invention will be further understood from the following illustrative and non-limitative examples of preferred embodiments thereof.

Brief description of the drawings

Fig. 1 graphically depicts the strong dose-dependent inhibition of hyaluronidase activity displayed by the Centella extract; Fig. 2 graphically depicts the synergy of action of Centella when added to Sambucus/Echinacea in a hyaluronidase inhibition model;

Fig. 3 graphically depicts the stimulatory effects on collagen synthesis in cultured fibroblasts upon the addition of three different herbal extracts/combinations;

Fig. 4 graphically depicts the stimulatory effects of the various herbal extracts on IL-6 release from cultured fibroblasts .

Detailed Description of Preferred Embodiments

As disclosed hereinabove, the present invention is primarily directed to the use of the herbal compositions described hereinabove in the preparation of medicaments for treating conditions characterized by degeneration of collagen and/or other extracellular matrix components.

The compositions comprising individual plant extracts or combinations thereof (as described above) may be administered to the subject being treated by any suitable route. In order to reduce the possibility of side effects, the compositions are generally given by local or topical administration.

Examples of suitable dosage forms for use in the presently disclosed method include (but are not limited to) liquids, gels, sprays, creams, ointments, pastes, lotions, solutions, and sustained release formulations. The compositions may also be formulated for administration by injection (via various routes including intradermal, subcutaneous, submucosal, intramuscular, intraarticular etc.). In one preferred embodiment the compositions may be incorporated into transmucosal or transdermal delivery systems. Examples of suitable transmucosal delivery systems may be found in co- owned international patent applications PCT/IL02/00402 (published as WO 02/094300) and PCT/IL2003/00159 (published as WO 04/047813) . Thus, in addition to the active ingredients, the compositions of the present invention may also contain one or more excipients including, but not limited to zinc, zinc oxide, silicones, calcium silicate, aluminum hydroxide, polyethylene glycols, methacrylate polymers and co-polymers, povidone derivatives, carbomers, magnesium stearate, fats of animal or vegetable origin, oils, waxes gums, starch and cellulose or cellulose derivatives. Further details regarding the manufacture of suitable formulations and the excipients used therein may be found in "Remington's Pharmaceutical Sciences", Mack Publishing Co, Easton, Pa, USA (1980) .

The compositions of the present invention are based on mixtures of plant extracts. It is to be noted that the term "extract" is used herein to include all of the many types of preparations containing some or all of the active ingredients found in the relevant plants. Thus the extracts may be produced by cold extraction techniques using a variety of different extraction solvents including, but not limited to, water, fatty solvents (such as olive oil), and alcoholic solvents (e.g. 70 % ethanol). Cold extraction techniques are usefully applied to softer parts of the plant such as leaves and flowers, or in cases wherein the desired active components of the plant are heat labile. Alternatively, the aforementioned solvents may be used to produce extracts of the desired plants by a hot extraction technique, wherein said solvents are heated to a high temperature, the precise value of said temperature being dependent on the properties of the chosen solvent, and maintained at that temperature throughout the extraction process. Hot extraction techniques are more commonly applied to the harder, tougher parts of the plant, such as bark, woody branches and larger roots. In some cases, sequential extractions need to be performed in more than one solvent, and at different temperatures.

Standard procedures for producing plant extracts (including hot extraction, cold extraction and other techniques) are described in many publications which are well known to the skilled artisan, one example of which is "Making plant medicine" [author: R. Cech, pub. by Horizon Herbs, 2000] .

As described above, compositions may be prepared using single extracts of either Sambucus nigra or Centella asiatica, or using a mixture of extracts of different plant species. In the latter case, the composition may be prepared using different ratios of each extract. For example, the aforementioned three-herb composition may comprise extracts of Centella asiatica, Echinacea purpurea and Sambucus nigra in the following range of percentage (weight/weight) ratios:

2%-60% : 0.5%-40% : 25%-99%

More preferably, these extracts are present in the percentage composition ratio of 20% : 10%: 70% In order to treat a patient with a composition containing either a single herbal extract or a mixture of herbal extracts as described hereinabove, it is necessary to administer said composition in a therapeutically-effective amount, that is, in an amount that will provide a concentration of the herbal extracts at the treatment site that is capable of exerting the desired therapeutic effect. It has been found, in general terms, that the compositions of the present invention need to be administered in amounts such that, typically, each topical dose contains between 0.1 mg and 10 mg (dry weight) of each herbal extract, the precise values depending on the particular combination of extracts used, and on the mode of topical delivery. Thus, by way of non-limiting example, in the case of the aforementioned three-herb composition of the present invention, the weights of the original plant material used to prepare a composition suitable for incorporation in a controlled-release delivery device are:

Centella asiatica 1.6 mg Echinacea purpurea 1.6 mg Sambucus nigra 7.56 mg

In the case of compositions intended primarily for topical use (e.g. on the skin for cosmetic use, to promote acclerated wound healing or - when placed above a joint - to treat degenerative joint disease), it is necessary to administer said compositions for a period of time that is sufficient to allow optimal contact of the therapeutically effective amounts of the herbal extracts with the lesions to be treated. When the compositions and medicaments are to be given by incorporation into a controlled-release device (such as hydrogel, dermal patches, sprays, creams or ointments) said device needs to remain in contact with the lesion (e.g. skin lesion) to be treated for a period of between 30 minutes and 5 hours. This treatment may be repeated up to 5 times each day, as required, and as determined by a competent clinician.

Hydrogels containing the compositions of the present invention should be taken in quantities of between 0.1 g and 15 g and allowed to remain in contact with the lesions to be treated for periods of between 1 minute and five hours. This treatment regime may be repeated up to 5 times per day.

Dermal patches, creams, ointments and other solid, soluble formulations are to be placed, if possible on the lesion or in close proximity to the lesions to be treated, and allowed to dissolve at the natural rate determined by the additives present in said formulations.

The following examples are provided for illustrative purposes and in order to more particularly explain and describe the present invention. The present invention, however, is not limited to the particular embodiments disclosed in these examples . EXAMPLES :

Example 1 Preparation of three-herb composition

The three-herb mixture mentioned hereinabove and used in the experimental investigations reported hereinbelow was prepared from a mixture of extracts of Centella asiatica, Echinacea purpurea and Sarnbucus nigra. This composition was prepared by mixing 6 ml of a 1:4 hydroalcoholic extract of Centella asiatica with 1.5 ml of a 1:1 hydroalcoholic extract of Echinacea purpurea and 35 ml of a 1:5 hydroalcoholic extract of Sambucus nigra. The weight ratio of C. asiatica : E. purpurea : S. nigra in this mixture is 15:15:70. The hydroalcoholic extracts of Centella asiatica and Echinacea purpurea were purchased from Herbal Apothecary, Syston, Leicester, U.K. or from Pentapharm, Basil, Switzerland, while the hydroalcoholic extract of Sambucus nigra was purchased from Analit Extracts Ltd., M. P. Hefer 38100, Israel, or from Pentapharm, Basil, Switzerland. It is be noted that the abovementioned extract values of the form l:x indicate that 1 g of the plant material was dissolved in x liters of solvent. The term "hydroalcoholic extract" indicates that the plant material was extracted using ethanol at concentrations of between 25% and 50% in water.

In the case of experimental investigations of single herb extracts the above hydroalcoholic extracts of Centella asiatica or Sambucus nigra were used in the biological assays alone . Example 2

Inhibition of hyaluronidase activity by an extract of Centella as±at±ca

Hyaluronic acid (also known as Hyaluronan) is a chief component of the extracellular matrix of connective tissue and epithelial and neural tissue. Hyaluronan is also a cell signaling molecule and has been strongly implicated in the healing of tissue. The inventors have now unexpectedly found that Centella asiatica extract possesses the ability to significantly inhibit the enzymatic degradation of hyaluronic acid by hyaluronidase. To measure the inhibition plastic wells were coated with human hyaluronic acid, the unbound sites on the wells blocked and the wells were then exposed to purified hyaluronidase in the absence or presence of various herbal extracts. Following incubation the wells were washed and incubated with a biotinylated peptide that binds to intact hyaluronic acid (i.e. not degraded by the enzyme). The plates were then developed by incubating with peroxidase conjugated to streptavidin followed by peroxidase substrate. As seen in Figure 1, Centella displays a strong dose dependent inhibition of hyaluronidase activity whereas Sambucus nigra, Echinacea purpurea, and Marshmallow root inhibit only weakly.

A comparison of the activity of equivalent doses of Centella with those found in the Sambucus/Centella/Echinacea mixture was then made, and it was found that addition of Centella to Sambucus/Echinacea caused an unexpected synergy in the inhibition of Hyaluronidase, as shown in Figure 2. The addition of the Marshmallow root to the Sambucus/Echinacea mixture in place of the Centella extract did not lead to any significant Hyaluronidase inhibition.

Example 3

Enhancement of fibroblast collagen production by a mixture of extracts of Sambucus nigra, Centella as±at±ca and Echinacea purpurea

Collagen production of confluent fibroblast monolayers was assessed by Sirius Red staining. The method is based on the interactions of anionic dye of Sirius Red with the side chain groups of the basic amino acids present in collagen molecule.

Fibroblasts were seeded in 24-well tissue culture plates in ImI of supplemented DMEM/10% FCS until confluence. Herbal extracts in ImI of DMEM, containing 0.1% BSA were added, and the cultures were incubated for 24 hr . The cell layers were extensively washed with PBS and fixed with ImI Bouin's fluid for lhr . The fluid was removed by suction and culture plates were washed in running tap water for 15 min . Then staining was done by addition of ImI of Sirius Red dye reagent for lhr under mild shaking on a micro plate shaker. In order to remove all non-bound dye, stained cell layers were extensively washed with 0.01 N hydrochloric acid, then the stained material was dissolved in 0.25 ml of 0.1 N sodium hydroxide under mild shaking for 30 min. Dye solution was transferred to Elisa 96- well plates and optical density was measured at 540 nm (using 0.1 N sodium hydroxide as a blank) . The stimulatory effects of the addition of three different herbal extracts/combinations on collagen synthesis by the cultured fibroblasts are summarized in Fig. 3, some of the details of which are explained in the following accompanying notes :

1. % over control (plotted on the Y-axis) was calculated as [OD treated^~0D untreated ) 1 00 ]

2. 105/8 + 49/26 = Sambucus/Echinacea combination

3. 7/10 = Centella

4. Ponaci = Sambucus/Echmacea/Centella combination

The results obtained are also summarized in the following table (mean + S.D.) :

It may be seen from the above results that the effect of combining the Centella extract with the Echinacea/Sambucus combination is much greater than additive. In other words, there is an unexpected synergistic interaction between Centella and the combination of the other two herbs on this model of collagen synthesis. Example 4

Enhancement of IL-1-induced IL-6 release by an extract of

Sambucus nigra alone, and by a composition containing Sambucus nigra, Centella asiatica and Echinacea purpurea

Gingival primary fibroblasts (passage 7) were seeded in 96- microwell tissue plates at a density of 10⁴ cell/well in 200μl of DMEM growth medium, containing 2mM-glutamine, 100 u/ml penicillin, lOOμg/ml streptomycin, (supplemented DMEM) and 10% heat-inactivated fetal calf serum (FCS). Cells were cultured at 37°C in a humidified atmosphere of 5% CO₂ overnight. Then the cells were stimulated with IL-lβ (5pg/ml) with or without the addition of herbal extracts at lmg/ml in DMEM supplemented with 0.1 % BSA. Cells were incubated for an additional 24 hours.

IL-I induced release of IL-6 was determined using a quantitative ELISA assay to measure IL-6.

The stimulatory effects of the addition of an extract of Sambucus nigra and (independently) of a composition containing a mixture of extracts of Sambucus nigra, Centella asiatica and Echinacea purpurea on IL-6 release by the cultured fibroblasts are summarized in the Fig. 4.

While specific embodiments of the invention have been described for the purpose of illustration, it will be understood that the invention may be carried out in practice by skilled persons with many modifications, variations and adaptations, without departing from its spirit or exceeding the scope of the claims.

Claims

1 A method for treating and/or preventing degeneration of collagen and/or other extracellular matrix components in a mammalian subject, wherein said method comprises administering to said subject a herbal composition comprising an extract of Centella asiatica and/or an extract of Sambucus nigra as the active component (s ).

2. The method according to claim 1, wherein the degeneration of collagen and/or other extracellular matrix components occurs as part of a condition selected from the group consisting of: degenerative joint diseases, keratoconjuctivitis and skin aging.

3. The method according to claim 1, wherein the other extracellular matrix component is hyaluronic acid.

4. The method according to claim 1, wherein the herbal composition comprises extracts of Centella asiatica and Samubucus nigra.

5. The method according to claim 4, wherein the herbal composition further comprises an extract of an Echinacea species .

6. The method according to claim 1, wherein the herbal composition comprises Centella asiatica as the sole active herbal component.

7. The method according to claim 1, wherein the herbal composition comprises Sambucus nigra as the sole active herbal component .

8. The method according to claim 1, wherein the herbal composition is administered to the subject by a route selected from the group consisting of intradermal, subcutaneous, submucosal, intramuscular, intraarticular and topical routes.

9. Use of a herbal composition comprising an extract of Centella asiatica and/or an extract of Sambucus nigra as the active component (s) in the preparation of a medicament for treating and/or preventing degeneration of collagen and/or other extracellular matrix components.

10. The use according to claim 9, wherein the degeneration of collagen and/or other extracellular matrix components occurs as part of a condition selected from the group consisting of degenerative joint diseases, keratoconjunctivitis and skin aging.

11. The use according to claim 9, wherein the other extracellular matrix component is hyaluronic acid.

12. Use of a herbal composition comprising an extract of Centella asiatica and/or an extract of Sambucus nigra as the active component (s) in the preparation of a cosmetic agent.

13. The use according to claim 9 or claim 12, wherein the herbal composition comprises extracts of Centella asiatica and Samubucus nigra.

14. The use according to claim 13, wherein the herbal composition further comprises an extract of an Echinacea species .

15. The use according to claim 9 or claim 12, wherein the herbal composition comprises Centella asiatica as the sole active herbal component.

16. The use according to claim 9 or claim 12, wherein the herbal composition comprises Sambucus nigra as the sole active herbal component.

17. A composition suitable for cosmetic use comprising an extract of Centella asiatica and/or an extract of Sambucus nigra as the cosmetically-active component ( s ).

18. The composition according to claim 17, comprising extracts of Centella asiatica and Samubucus nigra.

19. The composition according to claim 18, further comprising an extract of an Echinacea species .

20. A topical dosage form comprising the composition according to claim 17, wherein said dosage form is suitable for delivering the active ingredients of said composition to the dermal and sub-dermal tissues.